YANG Yan-mei ,LIU Pei ,LI Hong-mei ,PENG Huan ,DU Xia ,DONG Ye,HU Xian-qi

1 College of Plant Protection,Yunnan Agricultural University,Kunming 650201,P.R.China

2 State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan,Yunnan Agricultural University,Kunming 650201,P.R.China

3 Institute of Agricultural Environment &Resources,Yunnan Academy of Agricultural Science,Kunming 650205,P.R.China

4 International College,Yunnan Agricultural University,Kunming 650201,P.R.China

5 State Key Laboratory for Biology of Plant Disease and Insect Pests,Institute of Plant Protection,Chinese Academy of Agricultural Science,Beijing 100193,P.R.China

Abstract Meloidogyne vitis is a new root-knot nematode parasitic on grape root in Yunnan Province,China. In order to establish a rapid,reliable and specific molecular detection method for M.vitis,the species-specific primers were designed with rDNA-ITS (ribosomal DNA internal transcribed spacer) gene fragment as the target. The reaction system was optimized and the reliability,specificity and sensitivity of primer were testified,therefore,a rapid PCR detection method for M.vitis was established. The result showed that the optimal annealing temperature of the primers was 53°C,which was suitable for the detection of different life stages of M.vitis. Specificity test showed that the specific fragment size of 174 bp was obtained from M.vitis,but other five non-target nematodes did not have any amplification bands,thus effectively distinguish M.vitis and the other five species,and could specifically detect the M.vitis from mixed populations.Sensitivity test showed that this PCR technique could detect the DNA of a single second-stage juvenile (J2) and 10-4 female. Futhermore,this PCR technique could be used to detect directly M.vitis from soil samples. The rapid,sensitive and specific PCR molecular detection technique could be used for the direct identification of a single J2 of M.vitis and the detection of M.vitis in mixed nematode populations and the detection of two J2s or one male in 0.5 g soil samples,which will provide technical support for the investigation of the occurrence and damage of M.vitis and the formulation of efficient green control strategies.

Keywords: Meloidogyne vitis,PCR rapid detection,reliability,specificity,sensitivity

1.Introduction

Root-knot nematodes (RKNs) is a kind of highly specialized omnivorous plant pathogen with many species,wide host range,large damage area and serious economic loss. It has a wide range of species,and more than 100 species have been reported internationally (Jian 2011;Heet al.2020). RKNs poses a great threat to agricultural production,and crop damage generally results in yield reductions of 10-20% and up to 75% or more in worse situations (Weiet al.2005). Tobacco damage in Yunnan Province,China resulted in tobacco leaf losses of 20-30% and up to 50% in worse situations (Qinet al.1991). Kiwifruit damage in the main producing area of Guizhou Province reduced yield by 10-15% and up to 40% in worse situations (Taoet al.2017).

Vitis viniferaL.is an important fruit crop in China and has significant economic values. However,various pathogens,including plant-parasitic nematodes,seriously threaten the growth of grape,and RKNs is one of the important factors limiting grape production (Smithet al.2018). RKNs has been found to damage grape in North America,Asia,Europe,and Australia (Nicolet al.1999).Four common species of RKNs,Meloidogyne incognita,M.javanica,M.arenaria,andM.haplawere found to damage grape (Esmenjaud and Bouquet 2009). In addition,M.ethiopica,M.natalieiandM.thamesiare also reported to harm grape production (McLeod and Khair 1973;Goldenet al.1981;Carneiroet al.2007). The RKNs were found in grape in Linyi (Shandong Province),Luoyang,Zhengzhou,Kaifeng and Shangqiu (Henan Province),Dangshan and Xiaoxian (Anhui Province),and Huaihai Economic Zone (Jiangsu Province),wereM.incognita,and it is dominant population (Liet al.1991;Sun and Wang 1992;Yang 2003;Liuet al.2011;Liu and Zhang 2017).

The morphological identification centered on perineal patterns was the main method for early species identification of RKNs. However,morphological identification methods that rely on perineal patterns may face great challenges due to intra-species morphological variation and inter-species similarity in RKNs (Baidooet al.2016). Correaet al.(2014) and Carneiroet al.(2014) found thatM.incognitaandM.arenariaare similar toM.inornatain perineal patterns and are difficult to distinguish. Although isozyme electrophoresis technique is an important method for RKNs species identification because it is phenotypically stable and not easily influenced by environmental conditions,the technique does not enable the detection of second-stage juveniles(J2s),thus limiting its use for routine detection and diagnosis of J2s (Tiganoet al.2010). Molecular biology identification techniques can overcome the shortcomings of the above methods and gradually become an important method for RKNs species identification. DNA-based molecular biology methods have been widely used in the identification of a large number of RKNs species(Blok and Powers 2009). The ribosomal DNA-internal transcribed spacer (rDNA-ITS) of RKNs is one of the most commonly detection markers in the identification ofMeloidogynespp.(Htayet al.2016). Based on the sequence difference of rDNA-ITS ofDitylenchus arachis,Zhang (2016) developed a rapid detection technique forDitylenchus arachis. Htay (2017) designed a speciesspecific primers ofM.graminicolabased on rDNA-ITS sequence differences betweenM.graminicolaand other RKNs. According to the rDNA-ITS sequence difference ofM.panyuensis,the specific PCR detection technique ofM.panyuensiswas established (Heet al.2020). In addition,a variety of PCR-specific detection techniques for RKNs were developed,taking intergenic spacer region(IGS),28S-D2-D3 region and mtDNA region of ribosomal DNA of RKNs as target genes (Ji 2005;Liu 2005;Longet al.2006;Huang 2016).

Meloidogyne vitisis a new species of RKNs parasitic on grape root found in our previous study (Yanget al.2021).The nematode has high density in the infected area and caused serious damage to grapes,and it also has strong adaptive capacity,reproductive capacity and transmission capacity. Therefore,it is of great significance to develop a rapid,reliable and sensitive detection method for the prevention,quarantine and control ofM.vitis. Based on the rDNA-ITS sequence ofM.vitis,a species-specific primer were designed and screened in our previous studies (Yanget al.2021). In this study,the reliability,specificity and sensitivity of the primer were further tested,and a specific detection method was developed. This method has high specificity and sensitivity,which can be used for the detection ofM.vitisin single J2and the detection ofM.vitisin mixed nematode populations and soil samples.

2.Materials and methods

2.1.Nematode population samples

RKNs used in this study mainly includedM.vitis,M.incognita,M.javanica,M.arenaria,M.hapla,andM.enterolobii. The information was shown in the following Table 1.

Table 1 The nematodes information of Meloidogyne spp.

Females and eggs ofM.vitisboth were extracted from the root tissues of grapes. J2s ofM.vitiswere collected from hatching eggs,egg masses incubated in a constant temperature incubator at 28°C to collect the J2s. The egg masses were scattered with a dissecting needle,and males adherent in some egg masses were collected.

2.2.DNA extraction

DNA was extracted from a single female adult following the method described by Yanget al.(2020),whilethe extraction of DNA from single male,single J2,a large number of J2s and eggs were performed by the freeze-thaw method,as follows: Nematodes were picked and placed in a PCR tube containing 10 μL ddH2O,and 10 μL 1× PCR buffer (Mg2+plus) was added. Then,they were placed in a 95°C water bath kettle and liquid nitrogen for repeated freeze-thaw three times,5 min each time,0.5 μL proteinase K was added. After mixing,they were hold at 65°C for 15 min,95°C for 5 min,and centrifuged at 12 000 r min-1for 5 min.

2.3.rDNA-ITS sequence analysis and primers screening

Downloaded from GenBank,the sequences of the ITS region ofM.vitis(MN816222.1) and other nematodes species ofMeloidogynespp.,M.mail(KR535971.1),M.incognita(KJ641591.1),M.hapla(JX024147.1),M.arenaria(LC030350.1),M.javanica(JQ917440.1),M.enterolobii(JF309157.1),M.naasi(JN157859.1),andM.chitwoodi(JN241864.1). These sequences were aligned using DNAMAN and the most significantly different regions of the ITS region of these nematodes were selected for primer design using Primer Primer 5.0 Software,and the primers were screened with Oligo 6.0 Software. The obtained primers were synthesized by Beijing Liuhe Huada Gene Technology Company (China).

2.4.Primer reliability test

Detection of different life stage nematodesDNA was extracted from single female,J2,male,a large number of eggs for PCR amplification using the Mv-F/Mv-R primers shown in Table 2. PCR reaction system (25 μL): 10× PCR buffer (Mg2+plus) 2.5 μL,dNTPs (mixture) 2 μL,10 μmoL L-1forward and reverse primers 1 μL,respectively,DNA template 2.5 μL,5 U μL-1Taqpolymerase 0.25 μL,and ddH2O 15.75 μL. PCR amplification procedure is shown in Table 3. Amplification products were detected by 2%agarose gel electrophoresis and photographed in gel imager. The experiment was repeated three times,and water was used as the template for negative control.

Table 2 The primers information

Table 3 The PCR amplification procedure

Repeatability detectionDNA from 21 female adults ofM.vitis(21 replicates) was extracted,and the DNA of these 21 female adults were amplified by PCR to detect the repeatability of primers. The DNA extraction and PCR amplification procedure were performed as above,and water was used as a template for negative control.

2.5.Primer specificity test

DNA was extracted from six nematodes species populations ofM.vitis,M.incognita,M.javanica,M.arenaria,M.hapla,andM.enterolobii. The ITS sequences of the six nematode species were amplified by primer 18S/26S to confirm the existence of DNA.The specific primers of the six nematodes species were selected for specific amplification of the corresponding nematodes to ensure the accuracy of the selected nematodes. Finally,the specific primers Mv-F/Mv-R were used for PCR amplification of six nematode species. The experiment was repeated three times and water was used as the template of negative control.

2.6.Primer sensitivity test

Detection of single J2To detect primer sensitivity,different numbers of J2(n=1,2,3,4 and 5) were used to determine the minimum amount of J2detectable in the PCR assays. Then PCR amplification was performed using specific primers Mv-F/Mv-R. The experiment was repeated three times,and water was used as template of negative control.

Detection of different concentrations of DNA templateDNA was extracted from single female adult,and then the DNA was diluted in a 10-fold gradient,and the template of each concentration was amplified according to the PCR amplification procedure described in Table 3 to detect the sensitivity of the primers. The experiment was repeated three times,the water was used as template of negative control.

2.7.Detection of M.vitis in mixed populations

To further detect the reliability,specificity and sensitivity of the primers,one J2of each ofM.incognita,M.javanica,M.arenaria,M.hapla,M.enterolobii, andM.vitiswas selected and mixed for DNA extraction and PCR detection. Except for water,the DNA extracted from mixed nematodes (one J2ofM.incognita,M.javanica,M.arenaria,M.hapla,andM.enterolobii,respectively)was used as negative controls. The experiment was repeated three times.

2.8.Direct detection of M.vitis from soil sample

Seven pieces of 0.5 g sterilized soil were taken,different numbers of J2(n=1,2,4,6,8,and 10) and single male ofM.vitiswas added,respectively. After mixing,soil DNA was extracted and the DNA was used as a template for PCR amplification with Mv-F/Mv-R primers. The sterilization soil and water were used as template of negative control.The PCR amplification was performed using KOD FX DNA polymerase (TOYOBO,Osaka,Japan) containing 25 μL PCR reaction mixture: 2× PCR buffer for KOD FX 12.5 μL,2 mmol L-1dNTPs 5 μL,10 μmol L-1each primer 1 μL,DNA template 3 μL,1 U μL-1KOD FX DNA polymerase 1 μL,and ddH2O 1.5 μL. PCR amplification procedure:94°C for 2 min;40 cycles of 10 s at 98°C,30 s at 53°C,1 min at 68°C;hold at 4°C. A total of 5 μL amplification products were detected by 2% agarose gel electrophoresis.The experiment was repeated three times.

3.Results

3.1.Primer design result

Based on the results of rDNA-ITS sequence analysis,the specific primers Mv-F (forward:5´-CTGGTTCAGGGTCATTTATAAAC-3´),Mv-R (reverse:5´-TATACGCTTGTGTGGATGAC-3´) were designed between 5.8S and ITS2 regions ofM.vitis,which could specifically amplify 174 bp fragment fromM.vitis. The optimal annealing temperature of primer was 53°C,and the specific position of primer was shown in Fig.1-A and B.

3.2.Primer reliability test result

Detection result of different life stage nematodesAmplification of different life stages ofM.vitis(eggs,J2,female,and male) using the primers Mv-F/R revealed that the sequence sizes were 174 bp (Fig.2).

Repeatability detection result of primersAmplification of 21 single female adults ofM.vitisusing the primers Mv-F/R revealed that the specific sequence sizes were 174 bp(Fig.3).

3.3.Primer specificity test result

Amplification of the ITS fragment from six species nematodes revealed that the sequence size ofM.incognita,M.javanica,M.arenaria,M.hapla, andM.enterolobiiwere both about 760 bp,and 870 bp sequences size was obtained fromM.vitis,indicating that the DNA of all six nematodes was extracted successfully (Fig.4-A).

Based on the same DNA templates as mentioned above,specific bands of approximately 174,1 200,670,420,1 500,and 236 bp were amplified from the nematodes ofM.vitis,M.incognita,M.javanica,M.arenaria,M.hapla, andM.enterolobii, respectively,using six specific primer pairs of Mv-F/R,Finc/Rinc,Fjav/Rjav,Ma-F/R,Mh-F/R and Me-F/R,respectively (Fig.4-B).

Based on the same DNA templates ofM.vitis,M.incognita,M.javanica,M.arenaria,M.hapla, andM.enterolobiias mentioned above,the primers Mv-F/R were used for PCR amplification. The result showed that 174 bp specific bands was obtained fromM.vitis,while the other five RKNs and negative control did not show any amplified bands (Fig.4-C).

3.4.Primer sensitivity test result

Detection result of single J2The DNA of different numbers J2ofM.vitis(n=1,2,3,4,and 5) was used as template for PCR amplification and could obtain 174-bp sequence size,indicating that the primers Mv-F/R enabled to detect single J2ofM.vitis(Fig.5).

PCR amplification result of different concentrationsof DNA templatesThe DNA of single female adult was used as the initial template for continuous 10-fold gradient dilution to 10-5. PCR amplification results showed that the target bands could be amplified when the template concentration was diluted to 10-1,10-2,10-3,and 10-4,respectively. However,when the template was further diluted to 10-5,no any bands appeared,and no bands appeared in the blank control with water as the template(Fig.6). This indicates that the primers Mv-F/R are sensitive to detect DNA ofM.vitisup to 10-4female.

3.5.Detection of M.vitis in mixed populations

The DNA extracted from mixed nematodes populations of one J2of each ofM.incognita,M.javanica,M.arenaria,M.hapla,M.enterolobii,andM.vitiswas used as template,and specific bands of 174 bp were amplified by primers Mv-F/R. When there was noM.vitisin the mixed population,the specific primers Mv-F/R could not detect any band,and there was no band in the blank control with water as the template. This detection method could detectM.vitisin the mixed population (Fig.7).

3.6.Direct detection of M.vitis from soil sample

The amplification results showed that when one J2was added to 0.5 g soil,no bands could be detected. However,when two,four,six,eight,and ten J2s were added to 0.5 g soil,the specific target bands could be amplified. When one male was added to 0.5 g soil,the target bands could also be amplified. The specific primers could directly identify theM.vitisin soil,and the detection sensitivity was two J2s and one male in 0.5 g soil (Fig.8).

4.Discussion

Meloidogynevitisis a new species of RKNs parasitic on grapes found by our research group in Yunnan (Yang 2021). The high density ofM.vitisexists in the vineyards of the research area,which has posed a great threat to the local grape production and affected seriously the yield and quality of grapes. There are obvious differences in pathogenicity among different species of RKNs,therefore,accurate detection and identification of RKNs species is essential to study effective control scheme for RKNs (Chenet al.2013;Zhanget al.2014). However,studies on rapid identification methods forM.vitishave not been reported.

PCR detection technique is low cost,fast and efficient,and its reliability in RKNs species identification has been internationally recognized (Zijlstraet al.2000;Donget al.2001;Yanget al.2017). Sequence analysis revealed that the ITS regions ofM.vitiswas differed significantly from other RKNs,with 72.47% similarity to the closely related speciesM.mail,and 59.77,69.09,56.96,61.02,62.73,48.98,and 44.15% similarity to theM.incognita,M.hapla,M.arenaria,M.javanica,M.enterolobii,M.naasi,andM.chitwoodi,respectively. Based on the results of sequence analysis,the 5.8S-ITS2 region with the most significant difference from other RKNs was selected as the target,and a pair of specific primers Mv-F/Mv-R was designed and screened,which can achieve rapid identification ofM.vitis.

Reliability,specificity and sensitivity are important technical indicators of PCR-based detection methods. In terms of primer reliability,the primer Mv-F/R designed in this study was able to achieve detection of different life stages ofM.vitis. It was verified by sequencing that the primer could amplify sequences with 100% similarity between different life stages ofM.vitis. The main indicator of primer specificity is to detect the specific amplification of primers for target and non-target nematodes. Liu (2005)designed specific diagnostic primers forM.enterolobiiby selectingM.incognita,M.javanica,M.arenaria,M.hapla,andM.hispanicapopulations and mixed soil nematode populations as non-target samples to detect the specificity of the primers. When using LAMP technique to identifyM.enterolobii,Heet al.(2013) selected the non-target nematodes ofM.enterolobii,M.incognita,M.javanica,M.arenaria,M.hapla,Radopholus similis,Pratylenchus coffeae,Ditylenchus destructor,Heterodera glycines,Heterodera avenae,andBursaphelenchus xylophilusto detect the specificity of primers. In this study,M.incognita,M.javanica,M.arenaria,M.hapla,andM.enterolobiiwere used as non-target nematodes. Specific amplification results showed that specific bands were amplified only forM.vitis,and there were no amplified bands for non-target nematodes and water under the same amplification conditions. Primer sensitivity is also an important aspect of detection in SCAR-PCR technique. Longet al.(2006) and Tiganoet al.(2010)used successfully SCAR-PCR technique for detection of single RKN,Menget al.(2004) also used this technique to detect 1/3 J2of RKNs. Furthermore,the specific primers could directly identify theM.vitisin soil samples,which provides great convenience for the detection ofM.vitisfrom soil samples. The PCR detection system developed in this study can be used successfully for the detection of single J2and 10-4female ofM.vitis,and can realize the detection ofM.vitisin mixed populations.

Currently,the main specie of RKNs affecting grapes in China isM.incognita(Liet al.1991;Sun and Wang 1992;Yang 2003;Liuet al.2011;Liu and Zhang 2017).In this study,although just the five most common RKNs ofM.incognita,M.hapla,M.arenaria,M.javanica,andM.enterolobiiwere used for primer-specific detection and validation,the sample numbers of non-target nematodes was small with respect to the more than 100 RKNs reported so far. However,in the analysis of ITS sequence ofM.vitis,it is evident that there are significant differences even though they are morphologically and molecularly similar to other RKNs such asM.artiellia,M.floridensis,M.naasi,M.nataliei, andM.shunchangensis. Due to the lack of collected samples and the absence of domestic distribution reports of the related speciesM.mali,the specificity of the primer was not to detect withM.mali. However,as showen in Fig.1,there was a great difference in the base sequence of the primer design resigon (5.8S-ITS2 region) betweenM.vitisandM.mali. In the further detection,other RKNs,especially closely relatedM.maliand other species similar toM.vitisin morphology and molecular,should be collected and further detected and validated to determine the specificity of this primer.

5.Conclusion

In this study,we designed one species-specific primer pairs,named Mv-F (forward: 5´-CTGGTTCAGGGTCATTTA TAAAC-3´),Mv-R (reverse: 5´-TATACGCTTGTGTGGAT GAC-3´) based on the sequence of 5.8S-ITS2 region ofM.vitis,which could amplify specifically 174 bp fragment fromM.vitis. The primer pairs were highly specific and sensitive,and could be used for the direct identification of a single J2and 10-4female ofM.vitis,the detection ofM.vitisin mixed nematode populations and the detection of two J2s and one male in 0.5 g soil samples,which will provide technical support for the investigation of the occurrence and damage ofM.vitisand the formulation of efficient green control strategies.

Acknowledgements

This research was supported by the grants from the National Key Research and Development Program of China (2018YFD0201202 and 2017YFD0200601).

Declaration of competing interest

The authors declare that they have no conflict of interest.